B-cells participate in the removal of foreign antigens from the body by using a surface molecule to bind the antigen or by making specific antibodies that can search out and destroy specific foreign antigens. Each B-cell is specific for one antigen and will only produce antibodies against the same antigen that it bears the surface receptor for. However each B-cell can only make antibodies when it has bound its particular foreign antigen and received the appropriate approval signal from a T4 (helper T) cell. Once the T4 cell approval has been delivered to the B-cell the B-cell may continue producing antibodies as long as the foreign antigen is present.
In some cases the antibodies produced by the specific B-cells will inappropriately target host cells or antigens. For example, in certain autoimmune diseases, B-cells mistakenly make antibodies against tissues of the body (self antigens) instead of foreign antigens or make antibodies to foreign antigens that have enough similarity to self antigens to cause appropriately made antibodies to also react against tissues of the host's body. These auto-antibodies may interfere with the normal function of the tissues or initiate destruction of the tissues. For instance, people with myasthenia gravis experience muscle weakness because auto-antibodies produced by host B-cells attack a part of the nerve that stimulates muscle movements. Similarly, in the skin disease pemphigus vulgaris, auto-antibodies are misdirected against proteins that form the junctions between skin cells. The accumulation of these antibodies in between the skin layers causes severe skin blistering and death.
Other diseases in which antibodies mediate unwanted effects such as cell death include AIDS. AIDS has become a major cause of death in large parts of the world, with at least 40 million HIV infected individuals worldwide as of 2000 (U.S. Pat. No. 6,156,952). Recent improvements in therapy, such as the use of retroviral protease inhibitors, have been effective to decrease the mortality rates from AIDS. However, such therapies require continued use of expensive pharmaceuticals for the life of the infected individual. It appears that long-term use of such compounds may result in the development of resistant strains of HIV, as observed with earlier anti-HIV treatments like azidothymidine (AZT). Further, the expense of such treatments and demanding dosage regimens has prevented their widespread application in developing countries with some of the highest rates of HIV infection. A need exists for effective new therapies that can reduce or prevent the development of AIDS, preferably with a limited number or frequency of treatments.
It is almost universally agreed that AIDS is causally related to HIV infection, although the process by which HIV results in the immune system dysfunction characteristic of AIDS is still poorly understood. AIDS is characterized by a progressive depletion of CD4 positive immune cells (CD4+) from the host's system, including depletion of T4 helper/inducer cells (U.S. Pat. No. 5,767,072).
Early in HIV infection the immune response is largely cell-mediated, a few weeks after primary infection a vigorous HIV-specific cytotoxic T (T8) cell response develops and HIV levels decrease to nearly undetectable levels (Borrow et al., 1994; D'Souza and Mathieson, 1996; Koup et al., 1994). The initial antibodies produced in response to HIV infection are usually highly strain-specific and rapidly lose efficacy as the original HIV strain mutates (Koup et al., 1994; Albert et al., 1990; Tamalet et al., 1994). The high mutation rate intrinsic to retroviruses allows the HIV variants remaining in the host to escape immune surveillance and proliferate. For this reason, antibody therapy directed against HIV itself has not proven effective to date in preventing disease progression.
Following infection, the level of interleukin 2 (IL-2) production by T4 cells declines over a period of several years (Fan et al., 1993; Clerici and Shearer, 1993). There is a corresponding increase in levels of L-4 and IL-10, resulting in B cell activation and antibody overproduction (Graziosi et al., 1996). The resulting shift from a T8 and natural killer response to a B cell response is typical of individuals who will progress to full-blown AIDS (Fan et al., 1993; Clerici et al., 1993; Clerici et al., 1994; Meroni et al., 1996). This imbalance in interleukin production results in a feedback loop that further inhibits L-2 production as a result of increased L-10 levels (Groux et al., 1996). Decreased IL-2 production in turn contributes to the loss of T cell function (Chaplin, 1999).
One of the major antigenic determinants for host immune response to HIV is the gp120 glycoprotein which is the only HIV protein significantly exposed to the host's blood and lymph (Chaplin, 1999, Med. Hypoth. 52:133-146; U.S. Pat. No. 6,248,574). The gp120 protein is produced in precursor form by the env gene of HIV, which encodes a 160 kD glycoprotein (gp160) (U.S. Pat. No. 6,103,238). The gp160 protein is expressed in infected host cells and then cleaved into the extracellular surface protein gp120 and a transmembrane protein gp41. The transmembrane gp41 protein provides an anchor to which gp120 is somewhat loosely bound on the surface of HIV and infected host cells (Earl et al., 1991, J. Viro. 65:31-41).
Generally, the gp120 sequence can be divided into five variable regions (V1-V5) with 25% or less conserved sequences and five constant regions (C1-C5) with 75% or more sequence conservation, with immunologic determinants present on both constant and variable regions (Karry and Zouali, 1997). Variable regions V1-V4 form exposed loops anchored by disulphide bonds, while constant regions are concentrated in the core of the protein (Wyatt et al., 1998, Nature 393:705-711). The gp120 core, comprised of an inner domain, outer domain and a “bridging” anti-parallel β-sheet, has been reported to exhibit CD4 binding (Wyatt et al., 1998, Nature 393:705-711). Amino and carboxyl terminal sequences of gp120 are involved in gp41 binding (Wyatt et al., 1998, Nature 393:705-711).
Infection of T4 cells and macrophages by HIV is mediated by binding of gp120 to the CD4 receptor protein on the cell surface. (McDougal et al., 1986, Science 231:382-385). HIV is internalized into the cell and replicates by producing new viral genomes and viral proteins, including gp41 and gp120. New virions are produced by budding off from the infected cell membrane (Chaplin, 1999, Med. Hypoth. 52:133-146). About half of the gp120 protein produced is shed into the circulation, where it can bind to CD4 on non-infected T4 cells (Chaplin, 1999, Med. Hypoth. 52:133-146). Thus, gp120 in infected individuals may induce immune system attack on non-infected T4 cells as well as infected cells (Chaplin, 1999, Med. Hypoth. 52:133-146). This would account for the observation that the majority of T4 cells are eliminated by late-stage HIV infection, despite the fact that only about 1 in 10,000 T4 cells is infected early in HIV infection and only about 1 in 100 T4 cells is infected in terminal AIDS (Chaplin, 1999, Med. Hypoth. 52:133-146). Circulating anti-gp120 antibodies produced by activated B cells can bind to gp120 on the surface of infected and non-infected T4 cells, resulting in cross-linking and activation of antibody dependent cellular cytotoxicity (ADCC) directed against T4 cells (Chaplin, 1999, Med. Hypoth. 52:133-146).
The gp120 polypeptide includes a superantigen binding region as well as regions involved in binding to CD4 and to the chemokine coreceptor (CCR). In particular, glycosylated, unglycosylated and heat denatured forms of gp120 include a superantigen (SAg) region (including portions of V4 (e.g., residues 392-434) and C2 (e.g., residues 261-272)) that appears to interact with immunoglobulins of the VH3+ gene family in B cells (Karray and Zouali, 1997, Proc. Nat'l. Acad. Sci. USA 94:1356-1360, particularly FIG. 3; Goodglick et al., 1995, J. Immunol. 155:5151-5159; U.S. Pat. No. 5,691,135). In addition specific arginine residues in V3 loop of gp120 appear to be involved in binding of gp120 to the chemokine coreceptor (CXCR4 and CCR5) (Wang et al., 1998, P.N.A.S. 95:5740-5745 and Lin et al., 2001, J. Virol. 75:10766-10778). Furthermore, binding of gp120 to CD4 appears to involve many amino acids with Asp368, Glu370 and Trp427 being of particular importance (Wyatt et al., 1998, Nature 393:705-711).
The depletion of T cells in HIV patients appears to stem in part from B-cell production of antibodies against the envelope protein (e.g., gp120) of the HIV rather than by direct infection with HIV (Yang et al., 1996, J. Virol. 70:5799-5806). Indeed, between 80-100% of the cell death associated with HIV infection occurs in uninfected T4 cells (Weinhold et al., 1989, J. Immunol. 142:3091-3097; Finkel et al., 1995, Nat. Med. 1:129-134). It is believed that when gp120 is shed from the virus, the protein becomes either free-floating in the bloodstream or is bound to the surface of uninfected cells, particularly T4 cells via the CD4 receptors (Mittler and Hoffman, 1989, Science 245:1380-1382; Wang et al., 1994, Eur. J. Immunol. 24:1553-1557; Finco et al., 1997, Eur. J. Immunol. 27:1319-1324; Kang et al., 1997, Eur. J. Immunol., 28:2253-2264). Uninfected but dying T4 cells have been observed by numerous researchers to be coated with virally produced gp120 protein and anti-gp120 antibody. Additionally, multiple in vitro and in vivo model systems demonstrate AIDS-like immune system collapse with just these two components (Mittler and Hoffman, Wang et al., Finco et al., Kang et al.). Also, clinical retrospective studies have shown that progression to AIDS is strongly correlated with the combination of high gp120 levels and high anti-gp120 antibody concentration (Skowron et al., 1997, AIDS 11:1807-1814).
As the T4 cell death characteristic (and causative) of AIDS involves both virally produced gp120 and host produced anti-gp120 antibody, both of which are necessary and neither of which are sufficient, immunomodulatory therapies designed to eliminate the anti-gp120 antibody response may prove highly beneficial.
Immunosuppression therapy has been employed for the treatment of a number of diseases. Immunosuppression may be used for medical conditions in which a triggering event elicits an immune response, causing unwanted or deleterious responses in the host. Accordingly, immunosuppression finds use in autoimmune disease management, transplantation protocols, allergy management, and the like.
The term “autoimmune disease” as used herein is defined as a disorder that results from immune responses that directly or indirectly target host cells or tissue. Autoimmunity is an inappropriate and excessive response to self-antigens. Examples include but are not limited to, Graves' disease, Type I-Diabetes mellitus, pemphigus, autoimmune hepatitis, Rheumatoid arthritis, and Systemic lupus erythematous.
Drugs currently used to treat autoimmune diseases are non-specific immunosuppressive agents, such as anti-inflammatory agents or drugs which can block cell proliferation or depress proinflammatory cytokines, and, moreover, due to their non-specific immunosuppression effects, run counter to the goal of maintaining immune function in the presence of an HIV infection.
It is desirable to suppress the immune system in a more specific way to control the response to self-antigens and theoretically “cure” the disease without down-regulating the entire immune system. Several specific immunotherapies have been hypothesized and tested in recent years, many of which are impractical or do not work in humans.
A need exists for alternatives to general immunosuppression for the treatment of pathogen-induced autoimmune conditions such as HIV pathogenesis (AIDS) and antibody-mediated autoimmune diseases in general. Of particular interest would be the development of an immunosuppression protocol that is able to selectively eliminate a specific antigen-reactive B cell population of a host's immune system and prevent the mistargeting and destruction of health.
In an attempt to reduce or eliminate specific B-cells, several groups have attempted to introduce toxic antigens (e.g., ricin- or radio-labled antigens) that would destroy only B-cells producting that antibody. However, significant vascular leakage and non-specific cell necrosis was observed (Baluna, et al. (1999) P.N.A.S. 96:3957-3962, Baluna, et al. (1999) J. Immunotherapy 22:41-47, Soler-Rodriguez, et al. (1993) Exp. Cell Res. 206:227-234, and “Medical Aspects of Chemical and Biological Warfare” Office of the Surgeon General (1997) Chapter 32, Franz and Jaax). Fusions of antigen and toxin have also failed to exhibit the requisite specificity to be administered in the presence of a preexisting antibody response. (Ada et al. (1969) Nature 222:1291-1295.
Interaction of antibodies with traditional antigen-toxin fusions is a key concern as antigen bound antibodies are internalized by macrophages and dendritic cells and this uptake of the bound complex is expected to delete these and other key antigen-presenting components of the patient's immune system along with the targeted B cells. Given the extreme toxicity of these compounds it is imperative that they only target the intended B cell population, as a single misdirected drug molecule will kill a “bystander” cell. The present invention was designed as a two-component fusion toxin system with independent targeting mechanisms that must be correctly activated and overlap on the target cell population to deliver their two interdependent toxin moieties.